When specimens are examined under a microscope, there is often a need to reconstruct the three-dimensional shape of the surface of the object. Confocal scanning microscopy, for example, can be employed for this purpose. In such a case, a specimen is scanned point-by point with the focus of a light beam in a plane, so that an image is obtained in this image plane, although with only a small depth of focus. By recording several different planes and appropriately processing the images, the object can then be imaged three-dimensionally. Such a confocal scanning microscope method is known, for example, from U.S. Pat. No. 6,128,077. The optical components employed in confocal scanning microscopy, however, are very expensive and, in addition to sophisticated technical knowledge, they also require a great deal of adjustment work.
Furthermore, U.S. Pat. No. 6,055,097 discloses a method for luminescence optical microscopy. Here, a specimen is marked with dyes that are fluorescent under suitable illumination conditions, so that the dyes in the specimen can be localized by the irradiation. In order to generate a spatial image, a number of images are recorded in different focal planes. Each of these images contains image information stemming directly from the focal plane as well as image information stemming from spatial sections of the object that lie outside of the focal plane. For purposes of obtaining a sharp image, it is necessary to eliminate the image components that do not stem from the focal plane. In this context, the suggestion is made to provide the microscope with an optical system that allows the specimen to be illuminated with a special illumination field, for instance, a stationary wave, or with a non-periodic excitation field.
In order to improve the three-dimensional reconstruction of an object with a microscope, German patent application no. 100 50 963.0 proposes an improved method and an improved device. Here, an image-recording device, for example, a CCD camera, is used to record a series of images of an object in various z planes. Consequently, this yields a stack of plane images of the object from numerous different planes in the z direction of the object. Each of these images in the image stack contains areas of sharp image structures having a high sharpness of details and areas that were outside of the focal plane during the recording of the image and that are consequently present in the image in an unsharp state and without high sharpness of details. As a result, an image can be regarded as a set of partial image areas having different, especially high, sharpness of details and low sharpness of details. Image-analysis methods are then employed to extract the partial image areas from each image of the image stack, said areas being present in a high sharpness of details. Consequently, for each image of the image stack, a result image is obtained that now contains only the image areas of high sharpness. These result images are subsequently combined to form a new, detail-sharp three-dimensional overall image. The result is a new, completely detail-sharp three-dimensional microscopic image of the object.
Since the distances and the absolute positions of the z planes in which the images of the image stack were recorded are known, the three-dimensional microscopic image of the object thus obtained can also be evaluated quantitatively. Up until now, in order to record the individual images of the image stack in the various z planes, the distance between the lens and the object was changed by mechanically adjusting the height of the microscope stage, in other words, the stage on which the object rests. The high weight of the stage and the resultant inertia of the entire system, however, set a limit for the speed with which the individual image stacks can be recorded. After all, the inertia of the system means that a relatively long time is needed to move the object into the various z planes in the focus of the lens.